The present invention relates to devices and methods for the discrimination of particulate elements using flow analysis in walled conduits, and more particularly, to devices and to methods for optically discriminating particulate elements on the basis of different light scattering effects and different light emission behaviors stemming from structures (or substructures) of such elements as well as from differences in general compositional element characteristics.
Since establishment of the Cell Theory in the 1840""s clinicians have found it informative to categorize, analyze and monitor the diverse blood cell populations of man and beast. In the blood of any typical mammal these cells are the erythrocytes, leukocytes and thrombocytes. Their numerical concentration can be estimated with precision; provided however, sufficient cells are counted.
In health, mammals have five subpopulations of circulating leukocytes: neutrophil granulocytes, lymphocytes, monocytes, eosinophil granulocytes and basophil granulocytes. Since perfection of the light microscope around 1880 and the introduction of Ehrlich""s dyes for differentiating the granulocyte""s granules shortly thereafter, these leukocyte subsets can also be differentiated with precision; provided however, sufficient cells are examined. Additionally there is a need for rapid exact analysis of countless other suspendable complex micro-particles and cell types.
During last century it became clear from optic scattering effects and from other evidence that, even though proteins could not be resolved with classic microscopes and were in nanometer size ranges, these too must be considered micro-particles.
Whilst counting chambers and microscopes could be used for cell evaluations, investigators performing these tasks were prone to fatigue and subjective vagaries. Therefore, in relation to known statistical sampling errors, an inadequate number of micro-particles could be analyzed by microscopy and only extreme pathological deviations were intercepted reliably. On the other hand, by 1930 R. A. Fisher had taught that, if a sufficient number of well-chosen, different, robust measurements were made on complex organic entities such as cells during an investigation, then, it would be straightforward to objectively assign the different tested elements to appropriate natural sets and subsets.
From the 1930""s onwards attempts were made to automate the time-consuming number-accumulation chore and classification challenge of cell and micro-particle analysis. For clinical cell analysis, these endeavors evolved along two main paths: (i) image analysis microscopy, which examines all microscopic objects, and (ii) flow analysis, or generalized flow cytometry of micron-sized and of sub-micron particles down to macromolecules.
In classical microscopy a large specimen is placed into an object plane which is orthogonal to the optic axis of the microscope. That classical plane is occupied by a vast expanse of sample material which is illuminated over a wide field, i.e., a field much larger in area (and frequently also in depth) than the arbitrarily definable voxel (or volume element or xe2x80x9ccell of spacexe2x80x9d) occupied, for example, by a small single tissue cell or by an ensemble of granules within, perhaps, an eosinophil cell or a suspension or solution in a spectrophotometric cuvette. In whatever manner the voxel (or xe2x80x9ccellxe2x80x9d) is defined, there is a very vast number of these illuminated elements in the classical microscopic object. Each such illuminated voxel of such a sample preparation causes the scattering (or, in certain settings fluorescence) of light. In relation to the signal light from a specific voxel of interest, the light from all the other structures in the microscopic field represents signal-degrading background noise. That background noise light can easily exceed 99.9% of the total light received in the microscope""s image location of the specific voxel. Hence, significant detail relating to such classically viewed voxels is likely lost to the viewer.
xe2x80x9cConfocalxe2x80x9d microscopy is an automated technique for avoiding the degrading effects of that frequently overwhelming background noise discussed above. The background is avoided by contemporaneously having both the illumination lens system(s) and the interrogation lens system(s) focus essentially on one voxel at a time. Additionally, computer techniques are used to synthesize three-dimensional high-signal images whose rich information content could not otherwise be captured. Confocal microscopy was first patented in the United States in 1952 by Marvin Minsky. Today, such technique is practiced via instruments which matured in the 1980""s (see, for example, S. W. Paddock xe2x80x9cConfocal Laser Scanning Microscopyxe2x80x9d BioTechniques 27 992-1004 1999).
Thus, in the centuries-old field of microscopy the relatively recent introduction of the optic background-noise-reducing confocal microscopy technique has advanced the classic approach beyond illumination of the entire surrounding tissue to specific illumination and interrogation of optically-isolated elements of space, which can be made smaller than individual biologic cells. This lowers the limit of detection by increasing the signal-to-background ratio that can be differentiated as a separate element in the colloquial signal/noise function for any analyte or measurand. Without the confocal microscopy approach the sample signal is lost in the xe2x80x9cseaxe2x80x9d of system noise.
Returning now to the evolution of particle flow analysis, by the mid-century it has been said that considerable work had been done to produce instruments capable of counting blood cells. Interestingly, such instruments varied considerably in principle, mode of operation, complexity and cost.
Prior to the work of Crosland-Taylor in this area, optic flow cytometry evolved via the upright optic axis of the microscope with an object plane that was orthogonal to the optic axis. Crosland-Taylor made significant progress in overcoming the optic tissue background noise interference problem of microscopy by exploiting hydrodynamic forces within an optically clear suspension fluid to present biologic cells singly to an optic interrogation area in a so-called hydrodynamically focused cell monofile.
Throughout micro-particle analysis, one broad approach had been to suspend particulates in an appropriate fluid (e.g., as aerosol or hydrosol) in order to isolate a xe2x80x9csamplexe2x80x9d away from other interfering objects so that ensembles (down to single elements) could be interrogated in physically isolated elements of space. Specific sensors could then respond quantitatively to differing properties of the isolated elements. In this way multi-angle light scatter (MALS) studies had already allowed the interrogation of particle ensembles in the middle of cylindrical cuvettes (i.e., interrogation cells) far from the surrounding walls. The Crosland-Taylor work implemented a general approach to isolating thin, stable filaments of particulate-containing fluid for interrogation in flow-through cells rather than merely in static cells or cuvettes.
When lasers were introduced, MALS could more appropriately be termed MALLS (multi-angle laser light scatter); and in the 1960""s many scientists incorporated lasers into the earlier Crosland-Taylor flow cytometry systems. Thereby, additional optic sensing axes could be placed into the traditional orthogonal microscopic object plane which had been co-opted by flow cytometry for the particle monofile; and the illuminating laser pencil for flow cytometry could also reside in that plane. Indeed, many in the art perceived that an interrogation apparatus could be arranged with laser and particle filament intersecting at the optic scattering and fluorescence origin of a spherical radiation domain. Crosland-Taylor had already introduced bubble-free degassed carrying liquids. Therefore, except for the thin sample fluid filament carrying the test particulates through a finite illuminated radiation centroid, this spherical scattering space was occupied only by optically clear fluid in which the illuminating laser pencil need not leave a trace.
Such system enabled rapid MALLS examination of stained or unstained particles ranging in size from protein molecules through bacteria and mammalian cell organelles to large organic cells. Additionally, non-optic sensors such as electrical impedance and capacitance cell interrogation principles could be applied to each larger particle, whether concurrently with an optical interrogation or in exact tandem synchronizationxe2x80x94of a type which had evolved in the flow cytometry subfield of cell sorting (e.g., U.S. Pat. Nos. 3,710,933 and 3,989,381).
By the 1970""s flow cytometry could measure a host of both xe2x80x9cintrinsicxe2x80x9d and xe2x80x9cextrinsicxe2x80x9d particle properties. The so-called xe2x80x9cintrinsic propertiesxe2x80x9d can be documented without the use of special reagents while xe2x80x9cextrinsicxe2x80x9d properties are elicited via physical, chemical or biological reagents such as altered pH or tonicity and/or dyes and/or coupled monoclonal antibodies or tags.
FIGS. 1A and 1B summarize the state of flow analysis transducers in the 1970""s. These figures were based on work performed to produce vortex-assisted, super-focused, low-variance, low-noise, many-centimeter-long cell monofiles. In particular reference to the xe2x80x9ctankxe2x80x9d of FIG. 1A, U.S. Pat. No. 5,138,181 teaches that even without induction of a vortex xe2x80x9cthe fact that the positioning of the flow is provided by positioning the counting orifice, makes the alignment of the optical beam on the flow extremely stable.xe2x80x9d
Hydrodynamic focusing of the sample stream at the top results in a particulate filament, or monofile, which is stabilized by additional primary upstream and secondary downstream sheath inflows in both FIGS. 1A and 1B. This monofile jets through the impedance, laser, and other interrogation regions that are indicated schematically in the xe2x80x9ctankxe2x80x9d of FIG. 1A and about the xe2x80x9cconduitxe2x80x9d of FIG. 1B. That a secondary downstream sheath satisfied the Venturi needs of the jet not only in the xe2x80x9ctankxe2x80x9d, but, via fully stable countercurrent flow, also in the xe2x80x9cconduitxe2x80x9d was verified by the teachings of U.S. Pat. Nos. 4,515,274, 4,710,021, 5,378,633, 5,895,869, and 5,983,735.
From U.S. Pat. Nos. 4,710,021, 5,378.633, 5,895,869, and 5,983,735, it is taught that in many types of focused flow analysis the flow axes could be in any spatial direction. As shown in FIGS. 1C and 1D hereof, for bubble control the flow directions are generally arranged vertically upward when a compound transducer is not incorporated into systems that perform physical cell sorting. Using the stabilizing vortex lever Eisert also maintained very precisely located very thin monofiles in horizontal micrposcope object planes in what are essentially the xe2x80x9cflat chambersxe2x80x9d of familiar hemocytometers (see U.S. Pat. No. 4,110,043).
The dual structures of FIGS. 1A-1D symbolize a two decade schism in the sub-domain of flow analysis that exploited optic scatter:
1. scatter system flow analysis of sub-micron scatter structures in wall-illumination-avoiding fluid spaces, for example, the tank transducers of FIGS. 1A and 1C; and
2. scatter system flow analysis of supra-micron scatter structures in wall-illumination-degraded conduit bounded fluid spaces, for example, the conduit transducers of FIGS. 1B and 1D.
This dichotomy in scatter sensitivity did not extend to the thriving fluorescent applications of flow cytometry. Fluorescence system flow analysis of supra-micron scatter, fluorescent structures, and of sub-micron fluorescent (but not scatter) structures use conduit bounded fluid spaces such as that illustrated in FIGS. 1B and 1D. Of the sensors illustrated, they can take the form of either a coarse scatter sensor or a fluorescent sensor. If including a fluorescence sensor, then all incident-wavelength scatter noise (and scatter signal) is routinely filtered out from the longer emitted-wavelength measurements of these systems. With respect to fluorescent particle interrogations, these systems effectively reduce to that illustrated in FIG. 1C and do confocal fluorescent flow cytometry.
In the early 1970""s one of the desires of flow cytometry became recognition of xe2x80x9cintrinsicxe2x80x9d optic scatter of sub-microscopic structures such as bacteria, organelles and characteristics of small cells such as platelets. During this time, MALS explorations using helium-neon laser and argon ion laser Biophysics Cytograph systems with both circular and square cross-sections were conducted. However, when looking for the sub-microscopic scatter substructures known from electron microscopy (as opposed to also known fluorescent sub-micron substructures) it became evident that, despite the great improvements in hydrodynamic focusing made available per U.S. Pat. No. 3,871,770, there was a fundamental problem of signal-degrading optic scatter background noise in affordable walled transducer derivatives (e.g., FIG. 1B).
FIG. 1C shows diagrammatically that, in the wall-illumination-avoiding setting, micro-particles such as bacteria or cellular granules or macromolecules, do generate highly informative MALLS signatures which can be sampled over almost 4xcfx80 steradians about any angle outside the intense 13xc2x0 narrow-angle forward scatter cone.
Of course, the micro-particle illustrated in FIG. 1D also generates the same unique MALLS signals in the wall-illumination-degraded conduit structures. Unfortunately, using any of the available conventional flow cytometry illumination arrangements, the walls of these walled-conduit structures conventionally generates such an enormous quantity of background scatter noise, that, for practical purposes, the unique MALLS signatures from sub-micron structures can not be usefully discriminated therefrom. The improved positional, orientational and temporal variance that was enabled by spiral hydrodynamic focusing to overcome excitation field inhomogeneities was totally wasted in the optic scatter setting of walled-conduit flow cuvettes.
By the 1980""s Crosland-Taylor""s optic transducer flow cells had undergone numerous modifications and design variations (see, for example, U.S. Pat. No. 3,661,460). However, in each typical flow cell there had to be a physical boundary between the fluid medium carrying the particles and the media through which the illuminating light entered and left the fluid in the flow cell. Since these interfaces could never be optically perfect, they always gave rise to some light scatter noise. Rectangular conduits assembled from polished planar slabs could have optically quiet faces; however, the corner edges of those conduits always scattered the typically-used broad flat ribbons of illuminating laser light very intensely. With these ellipsoidal illumination ribbons, round walled conduits gave rise to enormous additional diffraction scatter degradations. Hence, after around 1980 practical attempts at collecting MALLS signals from regions intermediate between the orthogonal forward and lateral faces of classical rectangular flow cytometry flow cells of the type shown in FIGS. 1B and 1D were essentially abandoned.
Of all the focused illuminating light in such classical flow cytometry, far less than 1% will be intercepted by even a large particulate element of interest. Of such tiny intercepted fraction of light, far less than 1% will be scattered by the particle as signal light. If the particle is larger in diameter than the wavelength of the illuminating light, say a granulocyte examined with a helium-neon laser light, then far less than 1% of that scattered signal light is scattered in directions other than the narrow-angle forward direction (of an acceptance cone half-angle of, e.g., 13% from the optical axis of the laser).
As stated above, it has been known since around 1900 that particles of the order of size of the wavelength of the illuminating light, say the 300 nm to 700 nm eosinophilic granules within eosinophilic granulocytes, will scatter a relatively high proportion of characteristic signal light in the spatial region between said forward cone and the orthogonal or lateral direction. However, sub-micron particles additionally have a greatly reduced scattering efficiency with a scattering cross-section as low as 1% of geometrical cross-section around 100 nm (see U.S. Pat. No. 4,693,602). To this end, what became clear in the 1970""s in flow cytometry studies associated with U.S. Pat. No. 3,871,770 using walled-conduit flow cells was that, even if monochromatic laser light (rather than polychromatic mixed light) is used at around the wavelength of white light, then the signal for typical organic cells and microorganisms is almost totally degraded by scattered light from any close wall edges as in FIG 1D. Therefore sub-microscopic particles could not be analyzed sensitively in these classical flow cytometry flow cells by the MALLS techniques, which had been shown by some, theoretically and practically, to give the right discriminations; provided however, wall-illumination-avoiding analytical conditions are used.
Recapitulating, FIG. 1A shows a conventional wall-illumination-avoiding optic xe2x80x9ctankxe2x80x9d. In such an arrangement, hydrodynamically focused particulates generally pass from an impedance and/or capacitance or RF sensor into a fluid body which is so vast in relation to a particle file interrogated by a laser beam that optic irregularities at the very distant walls through which the laser enters and exits the fluid can be arranged to contribute mathematically negligible background noise to appropriate scatter signals gathered as in confocal microscopy at the illuminated center of a vast conceptual spherical radiation domain. FIG. 1B shows a wall-illumination-degraded optic xe2x80x9cconduitxe2x80x9d. In such a construction, hydrodynamically focused particulates generally pass from an impedance and/or capacitance or RF sensor into a transparent walled conduit flow cell. In such a typical clinical flow cytometry arrangement, the interrogated particle file is so close to the optically irregular flow cell walls that optic background noise scattered from those walls or edges overwhelms scatter signals from sub-micron particles. This happens not only when the conduit cross section is circular but also when that cross section is square or rectangular, for example, the cost-effective 250 xcexcmxc3x97250xcexcm (flow path dimensions), polished flow cells (Part No. 131.050-QS, Hellma, Corp.) which have been routinely available since the 1980""s.
This signal/noise scatter problem in walled-conduit flow cells was summarized in U.S. Pat. No. 4,515,274. The reference provides that, xe2x80x9cthe smaller the size of the flow cell 16, the better its optical characteristics, in that the flow cell approaches a point source for optical signals.xe2x80x9d As can be seen from this rationale, it is not conventionally the biological cell or micro-particle that is considered as the point source for the harvested optical signals, but the more than 10,000-fold larger illuminated flow cell. Unquestionably, the scatter signal from a biologic cell only rises with difficulty above the optic noise produced from such conventional systems. For more modern systems, this issue remains unresolved.
As opposed to addressing the generation of such background noise, enormous masking efforts and diffraction blocking is practiced in/by conventional systems to get the signal at the advocated and implemented single intermediate xe2x80x9cMALSxe2x80x9d scatter sensor to rise above the prevalent scatter noise. By contrast, in xe2x80x9ctanksxe2x80x9d without a wall-proximity and without that circular orifice diffraction scatter, the interception of innumerable low-noise MALLS signals has been systematically routine since the 1970""s.
To this end, an exemplary modern system is illustrated in U.S. Pat. 5,125,737. Such system, which is similar to that illustrated in FIG. 1D, does not teach any means for avoiding the generation of the overwhelming background noise scatter of FIG. 1D. Indeed, that patent essentially accepts as a conclusion that such background noise will be generated and that it is desirable to simply block out as much of the undesirable noise scatter as possible.
Since the 1940""s, it has been recognized that making optic measurements essentially simultaneously at differing scattering angles greatly assists in coping with the effect of inevitable degrading background noise. This is because, as in exploitation of the xe2x80x9ccommon mode rejection ratioxe2x80x9d technique which has long been used in electronic relationships, if a systematic error occurs in one measurement, and a basically similar systematic error is introduced in the coupled measurement, then the data can frequently be viewed in such a way that even large errors tend to cancel each other out. Thus, the measurements are no longer stochastically independent.
Formally, a xe2x80x9ccommon mode rejection ratioxe2x80x9d is measured in decibels and might here be defined as 20 log (I/xcex94i), wherein I is the absolute signal intensity at one selectably relevant robust broad annular scatter angle cone (e.g., at 13xc2x0), and xcex94i represents a difference between intensity values in certain measuring and reference scatter cones (e.g., a 13xc2x0 cone and a xe2x80x9cmissingxe2x80x9d MALLS cone). This second cone should not be the ubiquitous 90xc2x0 cone, as the scatter intensity has fallen off too greatly for supra-micron particle applications so far out. The second cone should be greater than 13xc2x0 but less than 90xc2x0; but classically, any arbitrarily selected single-angle cone in this region has been difficult to maintain from system to system when a full MALLS sensor series was not manufactured directly into one of the complex flow cells.
Accordingly, a need exists for a system having a walled-conduit that receives a particulate-carrying fluid filament and is subjected to an emitted laser beam for purposes of particulate discrimination to minimize light scatter background noise produced by an interaction of the walled-conduit and the laser beam. Moreover, a further need exists for such a system to provide sufficient reference and measurement scatter, produced from the laser beam-conduit interaction, to facilitate reliable application of the common mode rejection ratio technique.
At least one aspect of the present invention is drawn to a particle analyzing apparatus for discriminating particulate element(s) in a fluid flow for purposes of analysis, classification, sorting, and presentation. The apparatus has an illumination source for emitting an illumination beam, a flow cell, and a light sensor. The flow cell has: a flow passage, through which a fluid flow containing particles can pass, a first exterior face perpendicular to a first axis of the flow cell, and a second exterior face perpendicular to a second axis of the flow cell. Of note, the first axis is substantially co-axial with an illumination beam emitted from the illumination source, and the first exterior face and the second exterior face are orthogonal. The light sensor, oriented substantially parallel to the second exterior face of the flow cell and displaced a sufficient distance from the second axis to enable receipt of a prescribed range of light passed by the first exterior face, operatively receives light from the illumination source via the flow cell.
Another aspect of the present invention is directed to a particle analyzing apparatus for discriminating particulate element(s) in a fluid flow for purposes of analysis, classification, sorting, and presentation. The apparatus includes an illumination source to emit an illumination beam, a flow cell, a sample supply, and a first light sensor. The flow cell has: a flow passage extending through the flow cell, a rear surface portion that receives an illumination beam emitted by the illumination source, and a forward surface portion that passes light formed from an illumination beam received by the rear surface portion. The flow cell has a first axis, which is substantially co-axial with an axis of an illumination beam emitted from the illumination source, and a second axis, which is orthogonal to the first axis. The sample supply operates to introduce a fluid to the flow passage of the flow cell. The first light sensor operates to receive at least light passed from the flow cell in response to an interaction with an illumination beam emitted by the illumination source and a fluid supplied to the flow passage of the flow cell. A light receiving surface of the light sensor is substantially parallel to the axis of an illumination beam emitted from the illumination source, and the first light sensor is displaced a prescribed distance from the second axis.
Another aspect of the present invention is directed to a particle analyzing apparatus for discriminating particulate element(s) in a fluid flow for purposes of analysis, classification, sorting, and presentation. The apparatus has an illumination source to emit an illumination beam along an optical axis of the apparatus. Moreover, the apparatus includes a flow cell, a sample supply, and a light sensor. The flow cell, positioned along the optical axis, has a flow path extending through the flow cell as well as a portion of an exterior surface to receive an illumination beam emitted from the illumination source. The flow cell is oriented to prevent formation of back reflection, parallel to the optical axis, in response to an emission of an illumination beam from the illumination source. The sample supply functions to introduce a fluid to the flow path of the flow cell. The light sensor, positioned along the optical axis, operates to receive at least light passed from the flow cell in response to an interaction with an illumination beam emitted by the illumination source and a fluid supplied to the flow passage of the flow cell.
Another aspect of the present invention is directed to an optical system for a particle analyzing apparatus for discriminating particulate element(s) in a fluid flow for purposes of analysis, classification, sorting, and presentation. The apparatus includes an illumination source for emitting an illumination beam and at least one light sensor to receive at least a light component of an emitted illumination beam. The optical system includes a focusing optical system and a flow cell. The focusing optical system, positioned along an optical axis of the system, operates to receive an illumination beam emitted by the illumination source. The flow cell, positioned along an optical axis of the system and prior to the at least one light sensor, has: a flow passage, through which a fluid flow containing particles can pass, and an exterior surface portion to receive light passed by the focusing optical system. The surface portion of the flow cell, which operatively receives light passed by the focusing optical system, is not orthogonal to the optical axis of the system.
Another aspect of the present invention is directed to a particle analyzing apparatus for discriminating particulate element(s) in a fluid flow for purposes of analysis, classification, sorting, and presentation. The apparatus includes an illumination source to emit an illumination beam, a flow cell, a sample supply, a focusing optical system, and a light sensor. The flow cell has a flow passage extending through the flow cell, a rear surface portion that receives an illumination beam emitted by the illumination source, and a forward surface portion that passes light formed from an illumination beam received by the rear surface portion. The sample supply functions to introduce a fluid to the flow passage of the flow cell. The focusing optical system, positioned between the illumination source and the flow cell and having an aperture, forms and positions a beam waist of an emitted illumination beam relative to the flow passage. The first light sensor operates to receive light passed by the flow cell. The illumination beam source emits an illumination beam having an intensity profile characterized by a central, principal intensity peak that originates and terminates at substantially null intensity values. With respect to a synchronized relationship of elements of the system, the flow cell is further arranged relative to the illumination source so that the principal intensity peak is substantially centered with respect to the flow path, and such illumination beam intercepts internal boundaries of the flow cell, which define the flow path, proximate to the null intensity values.
An object of the present invention is to provide an optical system having a synchronized illumination beam and flow cell.
Another object of the present invention is to provide an optical system having a flow cell oriented to control back reflection when subjected to an illumination beam.
Another object of the present invention is to provide a particle analyzing apparatus, to discriminate particles in a fluid flow for analysis, classification, sorting, and presentation, having a flow cell-light sensor relationship to enable a gathering of a specific range of light scatter passed by the flow cell, such specific range of light scatter directly corresponding to at least one type of particle capable of being identified.
Another object of the present invention is address the objects of this invention, whether individually or in combination, and maintain an appropriate overall apparatus size.